Efficient propagation of adherently growing cells still represents an unsolved problem, despite the fact that a number of different approaches have been pursued in this regard (for an overview, see e.g. Partner et al. in Journal of Bioscience and Bioengineering, Vol. 100, No. 3, 235-245, (2005) and Warnock et al. in Biotechnol. Appl. Biochem., Vol. 45, 1-12 (2006)).
Propagation factors of only approx. 102, in relation to a batch procedure, also referred to as “passage” in cell culture technology, can be achieved with the best technical devices (fixed bed reactors, hollow fiber reactors) at the current time. However, significantly higher factors of 103 (e.g. for regenerative medicine and cell banks), 104 (e.g. production cells for biomolecules) up to 106 (e.g. for the food industry and agriculture) would be desirable.
This is precluded by the fact that adherent cells are only proliferative in a narrow density range. (Surface density approx. 500-50,000 cells/cm2). Outside of this density range, the cells do not grow or they die off.
A surface which increases in size with the cell count could help to remedy this. The problems associated with this approach have, however, not yet been technically solved. This is due on one hand to the fact the increase in surface size would have to follow an exponential growth, and on the other hand that the technical possibilities to realize growing surfaces have hitherto been greatly restricted.
The best possibility at the current time lies in the use of microcarriers. These are particulate bodies (often spherically shaped, but a disc and rod shape are also possible) of typically approx. 1 mm in diameter, on the surface of which the cells can grow. Nutrient supply is performed by a medium in which the microcarriers are suspended. As soon as the cells have entirely grown over the carrier, they are detached (generally enzymatically) and seeded to new carriers. The growth surface also increases with the number of carriers.
This approach has a number of disadvantages. Firstly, the reactor size must be adjusted to the number of carriers; the volume ratio of the carriers to the total reaction volume must be within certain boundaries (generally 25-50%). As a result of this, the use of one and the same reactor to achieve high expansion factors is prohibited; instead, there must be progression from smaller to larger reactors. Secondly, a process technique is required at all times which can handle solid bodies (microcarriers) in a mechanically gentle manner, which is significantly more complex in terms of equipment than dealing with liquids. Liquids can be conveyed, filtered, sterilized, etc. much more easily. Thirdly, the volumetric use of the reactor is not very efficient because the cells can only grow on the surface of the carriers. Fourthly, the cells on the microcarriers are exposed to shearing and impact forces which are generated by convection of the medium and collision of the carriers. Fifthly, analysis of the cells on the microcarriers is difficult. In general, the cells on the carriers do not lend themselves well to analysis by microscope. Sixthly, the supply of cells generally exclusively occurs through the medium, which must be provided in a volume-filling manner and as free of gradient as possible. This results in inefficient utilization of the medium. Seventhly, the seeding of cells to the carriers (inoculation) and also the harvesting of the cells from the carriers represent technically difficult processes in which large proportions of the cells are lost.
Against this background, one object of the invention is to provide improved means and methods for efficient cultivation of cells in adhesion culture with which the described advantages of the prior art can be avoided or at least significantly reduced.
It was possible to achieve this object by the provision of the cultivation method as well as the provision of the cell culture carrier according to the invention.